Difference between revisions of "Part:BBa K3733004"
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===Functional Parameters===
 
===Functional Parameters===
 
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To characterize this part, P<sub>phsA</sub> and PphsA151-342 were cloned into pSC101 vector separately. And they are all under the control of the two-component system ThsSR. We chose neGFP(https://parts.igem.org/Part:BBa_K3733012) as the reporter. Plasmids were transferred into <i>E. coli</i> DH5α. The strain was expanded in LB medium to OD=0.4, then 198μL of bacterial solution was spotted into a 96-well plate, and a series of concentration gradients (0mM, 0.001mM, 0.01mM, 0.1mM, 1mM, 10mM) of thiosulfate were added Sodium sulfate solution. Add 2μL of sodium thiosulfate to each well for induction and measure the fluorescence and OD600 in the Synergy H1 microplate reader overnight. In the experiment, the effect of the truncated promoter PphsA151-342 induced by thiosulfate does not change significantly compared with P<sub>phsA</sub>, which means that the deletion of the first 150 bp nucleotide of P<sub>phsA</sub> has no effects on the activity of the promoter. According to the description of Daeffler KN et al., the strength of the promoter after truncation will not increase significantly [1]. However, according to the results of our repeated experiments, the truncated promoter still slightly enhances the expression of downstream gene to a certain extent.
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To characterize this part, P<sub>phsA</sub> and P<sub>phsA151-342</sub> were cloned into pSC101 vector separately. And they are all under the control of the two-component system ThsSR. We chose neGFP(https://parts.igem.org/Part:BBa_K3733012) as the reporter. Plasmids were transferred into <i>E. coli</i> DH5α. The strain was expanded in LB medium to OD=0.4, then 198μL of bacterial solution was spotted into a 96-well plate, and a series of concentration gradients (0mM, 0.001mM, 0.01mM, 0.1mM, 1mM, 10mM) of thiosulfate were added Sodium sulfate solution. Add 2μL of sodium thiosulfate to each well for induction and measure the fluorescence and OD600 in the Synergy H1 microplate reader overnight. In the experiment, the effect of the truncated promoter PphsA151-342 induced by thiosulfate does not change significantly compared with P<sub>phsA</sub>, which means that the deletion of the first 150 bp nucleotide of P<sub>phsA</sub> has no effects on the activity of the promoter. According to the description of Daeffler KN et al., the strength of the promoter after truncation will not increase significantly [1]. However, according to the results of our repeated experiments, the truncated promoter still slightly enhances the expression of downstream gene to a certain extent.
 
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Revision as of 15:58, 17 October 2021


PphsA151-342

PphsA151-342 is a promoter under the control of the two-component system ThsSR, and it is improved from the PphsA(https://parts.igem.org/Part:BBa_K2507018) promoter. This promoter can be activated when the ThsSR two-component system senses high concentrations of thiosulfate.

Usage and Biology

PphsA is a promoter controlled by a two-component system consisting of ThsS(BBa_K2507000) and ThsR(BBa_K2507001). When ThsS feels thiosulfate, ThsR is phosphorylated, and phosphorylated ThsR can activate PphsA. And PphsA151-342 is a truncated promoter that retains PphsA nucleotides 151-342. Deleting the first 150 bp of the 342 bp PphsA nucleotide sequence has no effect on the promoter activation induced by thiosulfate, and it has a slight increase in the expression level of downstream gene.

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Figure 1. The nucleotide sequence of PphsA151-342 (the gray part is the deleted sequence of PphsA)


Functional Parameters

To characterize this part, PphsA and PphsA151-342 were cloned into pSC101 vector separately. And they are all under the control of the two-component system ThsSR. We chose neGFP(https://parts.igem.org/Part:BBa_K3733012) as the reporter. Plasmids were transferred into E. coli DH5α. The strain was expanded in LB medium to OD=0.4, then 198μL of bacterial solution was spotted into a 96-well plate, and a series of concentration gradients (0mM, 0.001mM, 0.01mM, 0.1mM, 1mM, 10mM) of thiosulfate were added Sodium sulfate solution. Add 2μL of sodium thiosulfate to each well for induction and measure the fluorescence and OD600 in the Synergy H1 microplate reader overnight. In the experiment, the effect of the truncated promoter PphsA151-342 induced by thiosulfate does not change significantly compared with PphsA, which means that the deletion of the first 150 bp nucleotide of PphsA has no effects on the activity of the promoter. According to the description of Daeffler KN et al., the strength of the promoter after truncation will not increase significantly [1]. However, according to the results of our repeated experiments, the truncated promoter still slightly enhances the expression of downstream gene to a certain extent. 无标题文档

Figure 2.Comparison of PphsA and PphsA151-342 induced by different concentrations of sodium thiosulfate

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Figure 3.The activation of PphsA(left) and PphsA151-342(right) under different induced concentrations of sodium thiosulfate

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference

<p> [1] Daeffler KN, Galley JD, Sheth RU, et al. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation[J]. Molecular systems biology, 2017, 13(4): 923.